This invention relates generally to the use of non-naturally occurring specific gold-binding proteins or peptides for use in analytic, exploration or recovery methods in the gold mining industry.
Gold is one of the rarest precious metals on earth. It occurs naturally as the reduced metal (Auxc2x0) or associated with quartz or pyrites as telluride (AuTe2), petzite (AuAg)2Te or sylvanite (AuAg)Te2. The electronics and space industries use gold""s properties of electrical conductivity and heat reflection. Gold has applications in radar equipment, home computers, satellites and space exploration. Gold is also used in considerable quantities in the form of gold leaf (having a thickness of less than 0.2 xcexcm) for sign writing and book binding lettering. Gold film has been used in glass windows to reflect heat. Liquid gold is a suspension of very finely divided gold particles in vegetable oil that is used in the decoration of china articles. Gold salts are used for toning in photography, and in coloring glass.
Most frequently gold in nature is dispersed in low concentration throughout large volumes of material, usually rock. Gold deposits occur in belts across the earth""s crust in various forms: placers or quartz veins in sedimentary or indigenous formation, blanket or pebble beds or conglomerates, or as base metal ore associations. Gold occurs in ore bodies described as lodes or veins, replacement deposits, contact (skarn) deposits, volcanogenic deposits, deposits associated with intrusive activity (such as xe2x80x98porphyryxe2x80x99 systems and breccia pipes) and deposits associated with ferruginous sediments (banded iron formations) and cherts. Gold bearing veins are found in rocks of all compositions and geologic ages, deposited in cavities and associated with rocks such as slates or schists. Lode deposits consist of gold particles contained in quartz veins or country rock. Lode deposits usually are mined in deep underground mines using a variety of methods, although sometimes lode deposits are surface mined. The blanket or reef-type deposits are deposits in which the gold exists in quartz conglomerates. Disseminated gold deposits have three identifying characteristics. The gold mineralization is fairly evenly distributed throughout the deposit rather than being concentrated in veins (as in lode deposits) or in pay-streaks (as in placer deposits); the deposits consist of in place materials rather than transported materials; and the disseminated deposits are less flat. Generally, these types of deposits are mined using surface mining techniques.
Gold also exists in secondary ore deposits. All rock outcrops exposed at the surface of the earth are subjected to the natural elements of weathering and erosion, causing eventual breakdown of rock into fragments which are carried away by wind, water or ice. The fragments are then redeposited in river systems, lakes or in the sea. During the erosive cycle, the heavier and more durable gold is concentrated into rich deposits, even though the original rock may have contained low values. Residual deposits of gold are found close to the gold bearing outcrop after the other rock fragments have weathered and been carried away. Eluvial deposits are formed when gold or gold bearing rock fragments have been transported short distances from their source (generally by gravity) and have been concentrated within the soil horizon. Alluvial deposits are formed by the concentration of gold particles within stream systems, under the action of running water. Beach placers, where gold is concentrated in beach sands by wave action, are a type of alluvial deposit. Leads are former stream courses, containing gold, where barren sands have covered the original passage of the stream. Deep leads are gold deposits in former stream beds which have been covered with basaltic lava. Nuggets are formed, either as rich fragments of primary deposits which have been transported and deposited in a sedimentary environment, or a chemical accretion of small gold particles into larger fragments. Some nuggets may have formed through the chemical action of host soils or sediments on a gold solution. Placer deposits are flat-laying deposits composed of unconsolidated materials, such as gravel and sands, in which the gold particles occur as free particles ranging in size from nuggets to fine flakes. They are the result of erosion and transport of rock. Placer deposits most commonly are mined using water based surface methods, including hydraulic methods, dredging and open pit mining. These deposits usually are not mined in underground operations.
Methods for recovering gold from its ores (termed xe2x80x9cbeneficiation methodsxe2x80x9d) are extremely expensive and labor and heavy machinery intensive. Gold is one of the least reactive metals on earth. It does not combine with oxygen or with nearly any other chemicals, no matter how corrosive. Some gold ores are free milling and allow the separation of coarse gold using methods that depend on the high specific gravity of gold. All other commonly used methods depend on the use of cyanide which is highly toxic, hazardous to the environment and difficult to remove. Basically, the first step in all methods is to subject the ore to cyanide leaching followed by a gold recovery process. The three known methods for extracting gold from the cyanide leach solution are the xe2x80x9cMerrill-Crowexe2x80x9d or zinc dust precipitation process, the carbon-in pulp process, and the carbon in-leach process. Other gold recovery processes use gravity methods to extract the high proportion of free gold and flotation-roasting leaching to extract the remaining gold.
Cyanide and cyanide by-products from cyanide leaching operations are responsible for several environmental impacts, including air and water pollution and solid waste disposal contamination. Free cyanide and various cyanide complexes are the by products of current leaching methods. Although cyanide will degrade, for example in a surface stream exposed to ultraviolet light, aeration and complexing with various chemicals present in the stream water, in-stream degradation is a wholly unsatisfactory approach to removing cyanide from the environment. Cyanide solutions are often kept in open ponds and frequently birds or other animals are exposed and killed by the toxic material.
Air pollution with cyanide also is an unavoidable result of prior art methods for heap-leaching of gold. Cyanide solutions are sprayed onto the heaps, the cyanide drifts and contaminates the surrounding environment. As is the case with cyanide released into water, eventually the cyanide is degraded by ultraviolet light, but not until after it has adversely affected the environment. The EPA directs considerable efforts and expense in regulating cyanide releases into the air and water. Chronic cyanide toxicity due to long-term exposures to low levels is also a health factor to be considered, and the effects such exposures are not presently well known. For these reasons there has been a long standing need for gold mining processes which do not pollute the environment with cyanide and cyanide byproducts.
Gold recovery from secondary sources such as electronic scrap and waste electroplating solutions, as well as recovery from primary sources such as leach solutions is also an important technology. Various processes such as carbon adsorption, ion exchange, membrane separation, precipitation, and solvent extraction have been used for isolation of metal ions, including gold.
Recently, methods for the utilization of naturally occurring proteins or biologic materials in analytic or gold recovery, including microbial biomass, as an adsorbent for metals have been studied. Bontideau et al., Anal. Chem. 70:1842-1848 (1997) is a physical chemical study of the two-dimensional binding properties between a naturally occurring protein and a gold substrate. The arrangement and enzymatic activity of a myosin sub-fragment were characterized with special focus on the direct attachment of the thiol groups of cysteines in the protein to the gold substrate.
The current process for gold recovery includes treatment with cyanide to form a gold cyanide complex. U.S. Pat. No. 5,378,437 of Kleid et al teaches the use of cyanide-secreting microorganisms that also absorb the cyanide gold complex once formed.
A large body of research exists that describes, as an alternative to cyanide, the utilization of biomass to recover gold from aqueous solution or suspension. U.S. Pat. No. 4,789,481 of Brierley describes an improvement over the basic biomass extraction process whereby the biomassxe2x80x94in this case Bacillus subtilisxe2x80x94is treated with a caustic solution prior to use. U.S. Pat. No. 4,769,223 of Volesky et al., is directed to the biomass process where the biomass is derived from the growth of the marine algae of the genus Sargassum. U.S. Pat. No. 5,567,316 of Spears et al., describes a process for recovering metals from solutions using an immobilized metalloprotein material. There is no suggestion that this process would be useful for the recovery or detection of gold.
Different processes of enrichment of gold-containing ore are known in the art. Flotation is one of the most widely used of these processes. In this method, separation is accomplished by treating ground ore with chemical reagents that cause one fraction to sink to the bottom of a body of water and the other fraction to adhere to air bubbles and rise to the top. The flotation process was developed on a commercial scale early in the 20th century to remove very fine mineral particles that formerly had gone to waste in gravity concentration plants. Most kinds of minerals require coating with a water repellent to make them float. By coating the minerals with small amounts of chemicals or oils, finely ground particles of the minerals remain unwetted and will thus adhere to air bubbles. The mineral particles are coated by agitating a pulp of ore, water, and suitable chemicals; the latter bind to the surface of the mineral particles and make them hydrophobic. The unwetted particles adhere to air bubbles and are carried to the upper surface of the pulp, where they enter the froth; the froth containing these particles can then be removed. Unwanted minerals that naturally resist wetting may be treated so that their surfaces will be wetted and they will sink. Processing the flotation concentrate in order to recover gold is simpler and cheaper than treatment of total ore stock. Current flotation technology, however, still does not recover all of the gold that is present, especially the gold in finely-dispersed ore. At least one attempt has been made to improve the flotation process using a microorganism culture. Cormack, et al., Gold Extraction Process for Bioflotation, WO 97/14818. In this method, a microorganism culture is introduced into flotation tails and the mixture is agitated.
Most reported research in the area of protein/gold interactions describes the adsorption of gold or other metals by proteins in a non-specific fashion. Ishikawa and Suyama, Recovery and Refining of Au by Gold-Cyanide Ion Biosorption Using Animal Fibrous Proteins, App. Biochem. and Biotech., 1998, 70-72:719-728, is typical. Animal fibrous proteins which were insoluble and stable in water, such as chicken feather protein and hen eggshell membrane, adsorbed gold in a non-specific fashion. In this reference, eggshell membrane was utilized in a column and was able to remove very low concentrations of gold from aqueous solution. Another typical reference which provides generic disclosure of protein/gold or protein/metal ion interactions is Alasheh and Duvnjak, Adsorption of Copper by Canola Meal, J. Hazardous Mat., 1996, 48:83-93. Niu and Volesky, Gold-cyanide Biosorption with L-cysteine, J. Chem. Tech, and Biotech., 2000, 75:436-442, describe the chelation properties of a particular amino acid. In this reference, biomass was xe2x80x9cloadedxe2x80x9d with L-cysteine by contacting dried, protonated biomass with a solution of L-cysteine, and resulted in the ability of the biomass to adsorb higher concentrations of gold-cyanide. The authors postulate that the enhanced binding probably results from binding the gold-cyanide complex to the cysteine NH3+, while the cysteine COOxe2x88x92 binds to positive charges on the biomass.
Brown, Nat. Biotech. 199715:269-72, herein incorporated by reference in its entirety, has engineered a fusion protein including E. coli alkaline phosphatase and an engineered gold binding peptide domain. The identification of the gold binding domain involved fusion of a combinatorial library of peptide repeat sequences to an outer membrane protein of E. coli. Cells were selected for their ability to attach to Au beads. The Au-binding domains that appeared to have high specificity and affinity for Au were then engineered as fusion peptides to the E. coli enzyme alkaline phosphatase (referred to as gold-binding protein or GBP). The attachment of the Au-binding domain to the enzyme provided a convenient means to follow (quantify) binding to Au surfaces. With respect to applications of this novel material, the article was principally concerned with studies on metal protein interactions. Woodbury et al., Biosensors and Bioelectronies, 13:1117-1126 (1998), is directed to the general application of the gold-binding peptides suggested by Brown. The biosensors described in the Woodbury et al. reference utilized the gold-binding peptides to attach recognition elements to the gold sensor surface. Detection of binding events to the recognition element is performed by surface plasmon resonance (SPR). Although the gold-binding peptide and its affinity to gold is an element of this article, the gold-binding peptides affinity to gold is not exploited for analytic or gold recovery applications.
The present invention provides methods for detecting gold in ore samples, using a gold-specific protein for binding to the sample. Such methods may be qualitative or quantitative. In one embodiment, the method is a direct-binding method. In another embodiment, the non-specifically bound gold-binding protein is proteolyzed and detected. The methods have been adapted for a high-throughput format, such as a multiwell plate.
The present invention also provides methods for extracting gold from a mineral suspension containing gold and magnetite, using a magnetite-binding gold-specific protein to form a magnetic complex and extracting gold using a magnetic field.
The present invention also provides flotation methods for extracting gold from a mineral suspension, using a gold-specific protein with a hydrophobic tail as a flotation reagent.